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Bone has multiple functions in animals, such as supporting the body for mobility. The zebrafish skeleton is composed of craniofacial and axial skeletons. It shares a physiological curvature and consists of a similar number of vertebrae as humans. Bone degeneration and malformations have been widely studied in zebrafish as human disease models. High-resolution imaging and different bone properties such as density and volume can be obtained using micro-computed tomography (micro-CT). This study aimed to understand the possible changes in the structure and bone mineral density (BMD) of the vertebrae and craniofacial skeleton with age (4, 12 and 24 months post fertilisation [mpf]) in zebrafish. Our data showed that the BMD in the vertebrae and specific craniofacial skeleton (mandibular arch, ceratohyal and ethmoid plate) of 12 and 24 mpf fish were higher than that of the 4 mpf fish. In addition, we found the age-dependent increase in BMD was not ubiquitously observed in facial bones, and such differences were not correlated with bone type. In summary, such additional information on the craniofacial skeleton could help in understanding bone development throughout the lifespan of zebrafish.

Mutations of the Gon4l/udu gene in different organisms give rise to diverse phenotypes. Although the effects of Gon4l/Udu in transcriptional regulation have been demonstrated, they cannot solely explain the observed characteristics among species. To further understand the function of Gon4l/Udu, we used yeast two-hybrid (Y2H) screening to identify interacting proteins in zebrafish and mouse systems, confirmed the interactions by co-immunoprecipitation assay, and found four novel Gon4l-interacting proteins: BRCA1 associated protein-1 (Bap1), DNA methyltransferase 1 (Dnmt1), Tho complex 1 (Thoc1, also known as Tho1 or HPR1), and Cryptochrome circadian regulator 3a (Cry3a). Furthermore, all known Gon4l/Udu-interacting proteins—as found in this study, in previous reports, and in online resources—were investigated by Phenotype Enrichment Analysis. The most enriched phenotypes identified include increased embryonic tissue cell apoptosis, embryonic lethality, increased T cell derived lymphoma incidence, decreased cell proliferation, chromosome instability, and abnormal dopamine level, characteristics that largely resemble those observed in reported Gon4l/udu mutant animals. Similar to the expression pattern of udu, those of bap1, dnmt1, thoc1, and cry3a are also found in the brain region and other tissues. Thus, these findings indicate novel mechanisms of Gon4l/Udu in regulating CpG methylation, histone expression/modification, DNA repair/genomic stability, and RNA binding/processing/export.

Interkinetic nuclear migration (INM) is a process by which nuclei oscillate between the basal and apical surfaces of epithelial cells in coordination with the cell cycle. The cytoskeletal machinery including microtubules and actin has been reported to drive apical INM; however, the role of nuclear proteins in this process has yet to be fully elucidated. Here, we investigated the function of a SUN-domain protein, Sun1, in zebrafish. We found that zebrafish sun1 is highly expressed in the ventricular zone of the brain. Knocking down sun1 with antisense morpholino oligonucleotides reduced the abundance of nestin- and gfap-expressing neural stem cells and progenitor cells. The live-cell imaging results showed that sun1 morphant cells migrated toward the basal side during the S phase but failed to migrate apically during the G2 phase. On the contrary, the passive stochastic movement during the G2 phase was unaffected. Furthermore, down regulation of sun1 was shown to reduce the expression of genes associated with the Notch pathway, whereas the expression of genes in the Wnt pathway was less perturbed. Findings from this research suggest that the Sun1-mediated nucleo-cytoskeletal interaction contributes to apical nuclear migration, and may thus affect exposure to Notch signal, thereby altering the composition of the progenitor pool in the embryonic neurogenesis of zebrafish.

To the Editor:We read with interest the article by Yang and colleagues.1 The authors argued that in their series of 29 families with RCBTB1 variants, none had FEVR syndrome, in contrast to what we ...

mibnn2002, identified from an allele screen, shows early segmentation defect and severe cell death phenotypes, which are different from those of other described mib mutant alleles. We have previously reported its defects in somitogenesis and identified its origin of mutation, a large deletion in LG2. The report here is a continuous study, where we applied the bioinformatics analysis to profile the genetic background of mibnn2002 mutants. By comparing the transcriptomic data of mibnn2002 mutants with those of AB wild-type, a total of 1945 differentially expressed genes were identified, including 685 up- and 1260 down-regulated genes. The Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis and Ingenuity Pathway Analysis (IPA) identified the enriched pathways and their related biological functions. Our data further demonstrated that the defects in the somitogenesis were related to the down-regulated segmentation genes, such as foxc1a, smyhc1, myod1 and mylpfa.

Treacher Collins syndrome (TCS) is a rare congenital birth disorder (1 in 50,000 live births) characterized by severe craniofacial defects. Recently, the authors' group unfolded the pathogenesis of polr1c Type 3 TCS by using the zebrafish model. Facial development depends on the neural crest cells, in which polr1c plays a role in regulating their expression. In this study, the authors aimed to identify the functional time window of polr1c in TCS by the use of photo-morpholino to restore the polr1c expression at different time points. Results suggested that the restoration of polr1c at 8 hours after fertilization could rescue the TCS facial malformation phenotype by correcting the neural crest cell expression, reducing the cell death, and normalizing the p53 mRNA expression level in the rescued morphants. However, such recovery could not be reproduced if the polr1c is restored after 30 hours after fertilization.

Although the zebrafish interrenal tissue has been used as a model for steroidogenesis and genesis of the adrenal gland, its specification and morphogenesis remains largely unclear. In the present study, we explored how the Wilms tumor 1 (WT1)-expressing cells are segregated from the SF-1-expressing steroidogenic cells in the zebrafish model. The interrenal tissue precursors expressing ff1b, the equivalent of mammalian SF-1, were derived from wt1-expressing pronephric primordia in the zebrafish embryo. Through histochemistry and in situ hybridization, we demonstrated that the size of functionally differentiated interrenal tissue was substantially increased on global inhibition of the Notch signaling pathway and was accompanied by a disrupted segregation between the wt1- and ff1b-expressing cells. As the Notch pathway was conditionally activated during interrenal specification, differentiation, but not ff1b expression, of interrenal tissue was drastically compromised. In embryos deficient for Notch ligands jagged 1b and 2b, transgenic reporter activity of wt1b promoter was detected within the steroidogenic interrenal tissue. In conclusion, our results indicate that Jagged-Notch signaling is required (1) for segregation between wt1-expressing cells and differentiated steroidogenic tissue; and (2) to modulate the extent of functional differentiation in the steroidogenic interrenal tissue.

Nasopharyngeal carcinoma (NPC) is a serious health problem in China and Southeast Asia. Relapse is the major cause of mortality, but mechanisms of relapse are mysterious. Epstein-Barr virus (EBV) reactivation and host genomic instability (GI) have correlated with NPC development. Previously, we reported that lytic early genes DNase and BALF3 induce genetic alterations and progressive malignancy in NPC cells, implying lytic proteins may be required for NPC relapse. In this study, we show that immediate early gene BRLF1 induces chromosome mis-segregation and genomic instability in the NPC cells. Similar phenomenon was also demonstrated in 293 and zebrafish embryonic cells. BRLF1 nuclear localization signal (NLS) mutant still induced genomic instability and inhibitor experiments revealed that BRLF1 interferes with chromosome segregation and induces genomic instability by activating Erk signaling. Furthermore, the chromosome aberrations and tumorigenic features of NPC cells were significantly increased with the rounds of BRLF1 expression, and these cells developed into larger tumor nodules in mice. Therefore, BRLF1 may be the important factor contributing to NPC relapse and targeting BRLF1 may benefit patients.

Skin depigmentation diseases, such as vitiligo, are pigmentation disorders that often destroy melanocytes. However, their pathological mechanisms remain unclear, and therefore, promising treatments or prevention has been lacking. Here, we demonstrate that a zebrafish insertional mutant showing a significant reduction of nicastrin transcript possesses melanosome maturation defect, Tyrosinase-dependent mitochondrial swelling, and melanophore cell death. The depigmentation phenotypes are proven to be a result of γ-secretase inactivation. Furthermore, live imaging demonstrates that macrophages are recruited to and can phagocytose melanophore debris. Thus, we characterize a potential zebrafish depigmentation disease model, a nicastrinhi1384 mutant, which can be used for further treatment or drug development of diseases related to skin depigmentation and/or inflammation.

Treacher Collins Syndrome (TCS) is a rare congenital birth disorder (1 in 50,000 live births) characterized by severe craniofacial defects, including the downward slanting palpebral fissures, hypoplasia of the facial bones, and cleft palate (CP). Over 90% of patients with TCS have a mutation in the TCOF1 gene. However, some patients exhibit mutations in two new causative genes, POLR1C and POLR1D, which encode subunits of RNA polymerases I and III, that affect ribosome biogenesis. In this study, we examine the role of POLR1C in TCS using zebrafish as a model system. Our data confirmed that polr1c is highly expressed in the facial region, and dysfunction of this gene by knockdown or knock-out resulted in mis-expression of neural crest cells during early development that leads to TCS phenotype. Next generation sequencing and bioinformatics analysis of the polr1c mutants further demonstrated the up-regulated p53 pathway and predicted skeletal disorders. Lastly, we partially rescued the TCS facial phenotype in the background of p53 mutants, which supported the hypothesis that POLR1C-dependent type 3 TCS is associated with the p53 pathway.

mib

Background Osmotic stress transcription factor 1/transforming growth factor-β-stimulated clone 22 domain 3 (Ostf1/Tsc22d3) is a transcription factor that plays an osmoregulatory role in euryhaline fishes. Its mRNA and protein levels are up-regulated under hyperosmotic stress. However, its osmoregulatory and developmental functions have not been studied in any stenohaline freshwater fishes. Zebrafish is an excellent model to perform such study to unfold the functional role of Tsc22d3. Methods We identified the zebrafish Tsc22d3 and performed knockdown studies using morpholino antisense oligonucleotide (MO). Results Zebrafish Tsc22d3 did not response to hypertonic stress and ts22d3 knockdown or overexpression by injecting MO or capped RNA did not change the transcriptional levels of any of the known ionocyte markers. To reveal the unknown function of zebrafish Tsc22d3, we performed several in situ molecular marker studies on tsc22d3 morphants and found that Tsc22d3 plays multi-functional roles in dorsoventral (DV) patterning, segmentation, and brain development. We then aimed to identify the mechanism of Tsc22d3 in the earliest stages of DV patterning. Our results demonstrated that tsc22d3 is a ventralizing gene that can stimulate the transcription of bone morphogenetic protein 4 ( bmp4 ) and, thus, has a positive effect on the Bmp signaling pathway. Furthermore, we showed that Tsc22d3 interacts with deubiquitylating enzymes, ubiquitin-specific protease 15 (Usp15) and ovarian tumor domain containing protein 4 (Otud4). In addition, the interruption of Bmp4 signaling by double knockdown of usp15 and otud4 reduced the ventralized effects in tsc22d3 -overexpressing embryos. Conclusions This is the first study to identify new developmental functions of Tsc22d3 in zebrafish. General significance Zebrafish tsc22d3 is a ventralizing gene and plays a role in early embryogenesis.

Rationale: Hypoxia is a significant perturbation that exacerbates endothelial barrier dysfunction, contributing to the disruption of vascular homeostasis and the development of various diseases such as atherosclerosis and metastasis of tumors. To date, it is not known what strategy might be used to counter the effect of hypoxia on endothelial permeability. Objective: This study investigated the role of nitrite in regulating vascular integrity under hypoxic conditions. Methods and Results: We found denitrosylation and the resulting activation of caspase-3 to be critical for hypoxia-induced endothelial permeability. Nitrite treatment led to S -nitrosylation and the inactivation of caspase-3, suppressing the barrier dysfunction of endothelia caused by hypoxia. This process required the conversion of nitrite to bioactive nitric oxide in a nitrite reductase-dependent manner. Using primary human umbilical vein endothelial cells as a model, we showed that in the presence of nitrite, the S -nitrosylated and inactivated form of caspase-3 was unable to cleave β-catenin, a key component in the VE-cadherin complex. Therefore, nitrite treatment led to the maintenance of VE-cadherin–mediated adherens junctions under hypoxic conditions. In in vivo experiments using a zebrafish model, nitrite was found to protect blood vessels from hypoxia-induced vascular leakage. Conclusions: These results are the first to demonstrate that nitrite plays a critical role in the protection of endothelial barrier function against hypoxic insult. Our findings show that nitrite holds great potential for the treatment of diseases associated with hypoxia-induced disorder of vascular homeostasis.

Somites are formed progressively from the presomitic mesoderm (PSM) in a highly regulated process according to a strict periodicity driven by an oscillatory mechanism. The Notch and Wnt pathways are key components in the regulation of this somitic oscillator and data from Xenopus and zebrafish embryos indicate that the Notch-downstream target Nrarp participates in the regulation of both activities. We have analyzed Nrarp/nrarp-a expression in the PSM of chick, mouse and zebrafish embryos, and we show that it cycles in synchrony with other Notch regulated cyclic genes. In the mouse its transcription is both Wnt- and Notch-dependent, whereas in the chick and fish embryo it is simply Notch-dependent. Despite oscillating mRNA levels, Nrarp protein does not oscillate in the PSM. Finally, neither gain nor loss of Nrarp function interferes with the normal expression of Notch-related cyclic genes.

Summary Recent studies have begun to elucidate how the endothelial lineage is specified from the nascent mesoderm [1, 2]. However, the molecular mechanisms which regulate this process remain largely unknown. We hypothesized that Notch signaling might play an important role in specifying endothelial progenitors from the mesoderm, given that this pathway acts as a bipotential cell-fate switch on equipotent progenitor populations in other settings [3, 4]. We found that zebrafish embryos with decreased levels of Notch signaling exhibited a significant increase in the number of endothelial cells, whereas embryos with increased levels of Notch signaling displayed a reduced number of endothelial cells. Interestingly, there is a concomitant gain of endothelial cells and loss of erythrocytes in embryos with decreased Notch activity, without an effect on cell proliferation or apoptosis. Lineage-tracing analyses indicate that the ectopic endothelial cells in embryos with decreased Notch activity originate from mesodermal cells that normally produce erythrocyte progenitors. Taken together, our data suggest that Notch signaling negatively regulates the number of endothelial cells by limiting the number of endothelial progenitors within the mesoderm, probably functioning as a cell-fate switch between the endothelial and the hematopoietic lineages.

In vertebrates, hindbrain is subdivided into seven segments termed rhombomeres and the interface between each rhombomere forms the boundary. Similar to the D/V boundary formation in Drosophila, Notch activation has been shown to regulate the segregation of rhombomere boundary cells. Here we further explored the function of Notch signaling in the formation of rhombomere boundaries. By using bodipy ceramide cell-labeling technique, we found that the hindbrain boundary is formed initially in mib mutants but lost after 24 hours post-fertilization (hpf). This phenotype was more severe in mibta52b allele than in mibtfi91 allele. Similarly, injection of su(h)-MO led to boundary defects in a dosage-dependent manner. Boundary cells were recovered in mibta52b mutants in the hdac1-deficient background, where neurogenesis is inhibited. Furthermore, boundary cells lost sensitivity to reduced Notch activation from 15 somite stage onwards. We also showed that knockdown of notch3 function in notch1a mutants leads to the loss of rhombomere boundary cells and causes neuronal hyperplasia, indicating that Notch1a and Notch3 play a redundant role in the maintenance of rhombomere boundary. Electronic supplementary material The online version of this article (doi:10.1007/s00427-009-0296-6) contains supplementary material, which is available to authorized users.

High-throughput screening with a loss-of-function strategy is a logical and efficient way to identify novel genes involved in biological processes of interest. In zebrafish, morpholinos have been developed as a convenient tool to knock down gene expression. Here, we describe procedures for systematic screening using morpholinos in zebrafish to identify novel deubiquitylases involved in convergent extension during gastrulation. In this example, we examine candidates based on embryonic morphology and molecular signals of whole mount in situ hybridization assay.

Both mind bomb ( mib ) and mind bomb-2 ( mib2 ) encode RING E3 ubiquitin ligases that promote Delta ubiquitylation and endocytosis in Notch activation. Detailed morphological and molecular examinations revealed that zebrafish mib ta52b (missense mutation in the C-terminal RING Finger (RF), M1013R) and mib m132 (nonsense mutation resulting in a truncated protein that loses all three RFs, C785stop) are strong and weak antimorphic alleles, respectively, compared to the null allele, mib tfi91 (nonsense mutation resulting in a truncated protein of only 60 amino acids, Y60stop). Zebrafish mib2 ortholog was identified in this study. Zebrafish Mib and Mib2 are colocalized in transfected cells and function redundantly in regulating Notch signaling in embryos. Mib ta52b and Mib m132 have a dosage-dependent dominant-negative effect, at least, on Mib2, which is a molecular basis for the antimorphic phenotypes. It was also shown that Notch signaling negatively regulates mib expression in a Su(H)-dependent manner, forming a negative feedback loop in modulating Notch activation.

It was already known that both mind bomb (mib) and mind bomb-2 (mib2) encode E3 ubiquitin ligases that target Delta in Notch activation. Here we further demonstrated that zebrafish Mib and Mib2, similar to their mouse orthologs, have a C-terminal-most RING finger-dependent E3 ubiquitin ligase activity. Mib and Mib2 are reciprocal E3 ubiquitin ligases and substrates. They function similarly in Notch signaling by using DeltaC as a common substrate. However, Mib2 behaves differently from Mib in DeltaD internalization. In addition, Mib and Mib2 bind differently to extracellular and intracellular parts of DeltaA and DeltaC. Finally, mutant Mibs, Mib(ta52b) with a missense mutation in the C-terminal-most RING finger (M1013R) and Mib(m132) with a premature stop codon that leads to a deletion of three RING fingers (C785stop), act dominant-negatively and compete with Mib2 in DeltaC ubiquitylation and internalization, suggesting a molecular basis for the antimorphic phenotypes (stronger than the null phenotypes) observed in zebrafish mib(ta52b) and mib(m132) alleles.

Familial exudative vitreoretinopathy (FEVR) belongs to a group of genetically and clinically heterogeneous disorders in retinal vascular development. To date, in approximately 50% of patients with FEVR, pathogenic mutations have been detected in FZD4, LRP5, TSPAN12, NDP and ZNF408. In this study, we identified two heterozygous frameshift mutations in RCBTB1 from three Taiwanese cases through exome sequencing. In patient-derived lymphoblastoid cell lines (LCLs), the protein level of RCBTB1 is approximately half that of unaffected control LCLs, which is indicative of a haploinsufficiency mechanism. By employing transient transfection and reporter assays for the transcriptional activity of β-catenin, we demonstrated that RCBTB1 participates in the Norrin/FZD4 signaling pathway and that knockdown of RCBTB1 by shRNA significantly reduced nuclear accumulation of β-catenin under Norrin and Wnt3a treatments. Furthermore, transgenic fli1:EGFP zebrafish with rcbtb1 knockdown exhibited anomalies in intersegmental and intraocular vessels. These results strongly support that reduced RCBTB1 expression may lead to defects in angiogenesis through the Norrin-dependent Wnt pathway, and that RCBTB1 is a putative genetic cause of vitreoretinopathies.

mibnn2002, found from an allele screen, showed early segmentation defect and severe cell death phenotypes, which are different from previously known mib mutants. Despite distinct morphological phenotypes, the typical mib molecular phenotypes: her4 down-regulation, neurogenic phenotype and cold sensitive dlc expression pattern, still remained. The linkage analysis also indicated that mibnn2002 is a new mib allele. Failure of specification in anterior 7-10 somites is likely due to lack of foxc1a expression in mibnn2002 homozygotes. Somites and somite markers gradually appeared after 7-10 somite stage, suggesting that foxc1a is only essential for the formation of anterior 7-10 somites. Apoptosis began around 16-somite stage with p53 up-regulation. To find the possible links of mib, foxc1a and apoptosis, transcriptome analysis was employed. About 140 genes, including wnt3a, foxc1a and mib, were not detected in the homozygotes. Overexpression of foxc1a mRNA in mibnn2002 homozygotes partially rescued the anterior somite specification. In the process of characterizing mibnn2002 mutation, we integrated the scaffolds containing mib locus into chromosome 2 (or linkage group 2, LG2) based on synteny comparison and transcriptome results. Genomic PCR analysis further supported the conclusion and showed that mibnn2002 has a chromosomal deletion with the size of about 9.6 Mbp.

Borders are essential for demarcating repeated structures such as somites during vertebrate development. Two recent articles describe roles for Integrinalpha5 and its ligand Fibronectin1 in zebrafish anterior intersomitic boundary formation and link them to Notch and Eph-Ephrin pathways in epithelialization of somite boundary cells. Together with these pathways, Integrinalpha5 and Fibronectin1 orchestrate the orderly formation of somite and later myotome borders. These studies shed light on components downstream of the periodic segmentation mechanism - the 'segmentation clock' - in somitogenesis.

The formation of localised signalling centres is essential for patterning of a number of tissues during development. Previous work has revealed that a distinct population of boundary cells forms at the interface of segments in the vertebrate hindbrain, but the role of these cells is not known. We have investigated the function of the Wnt1 signalling molecule that is expressed by boundary and roof plate cells in the zebrafish hindbrain. Knockdown of wnt1 or of tcf3b, a mediator of Wnt signalling, leads to ectopic expression of boundary cell markers, rfng and foxb1.2, in non-boundary regions of the hindbrain. Ectopic boundary marker expression also occurs following knockdown of rfng, a modulator of Notch signalling required for wnt1 expression at hindbrain boundaries. We show that the boundary and roof plate expression of wnt1 each contribute to upregulation of proneural and deltagene expression and neurogenesis in non-boundary regions, which in turn blocks ectopic boundary marker expression. Boundary cells therefore play a key role in the regulation of cell differentiation in the zebrafish hindbrain. The network of genes underlying the regulation of neurogenesis and lateral inhibition of boundary cell formation by Wnt1 has a striking similarity to mechanisms at the dorsoventral boundary in the Drosophila wing imaginal disc.

Drosophila fringe and its homologues in vertebrates code for glycosyltransferases that modify Notch, altering the sensitivity of this receptor protein to its ligands Delta and Serrate and, thereby, playing an essential part in the demarcation of tissue boundaries. We describe the isolation and characterization of three zebrafish (Danio rerio) fringe homologues: lunatic fringe (lfng), radical fringe (rfng), and manic fringe (mfng). In addition to the sites previously described (Prince et al. [2001] Mech. Dev. 105:175–180; Leve et al. [ 2001] Dev. Genes Evol. 211:493–500), lfng is also expressed in the sensory patches of the inner ear. The newly described rfng is expressed in adaxial cells, tectum, rhombomere boundaries, and formed somites, but the expression of mfng is only detectable by reverse transcription-polymerase chain reaction and not by whole-mount in situ hybridization (WISH) during early embryonic development; later, it is expressed in the sensory patches of the ear. In mib mutants, where Notch signaling is defective and rhombomere boundaries fail to form, the rfng expression in hindbrain is almost completely lost. None of the three zebrafish fringe genes is detectably expressed in the posterior presomitic mesoderm, suggesting that, in contrast with chick and mouse, the somitogenesis oscillator in this tissue in the zebrafish does not depend on Fringe activity. Developmental Dynamics 231:621–630, 2004. © 2004 Wiley-Liss, Inc.

Vertebrate segmentation is manifested during embryonic development as serially repeated units termed somites that give rise to vertebrae, ribs, skeletal muscle and dermis. Many theoretical models including the “clock and wavefront” model have been proposed. There is compelling genetic evidence showing that Notch–Delta signaling is indispensable for somitogenesis. Notch receptor and its target genes, Hairy/E(spl) homologues, are known to be crucial for the ticking of the segmentation clock. Through the work done in mouse, chick, Xenopus and zebrafish, an oscillator operated by cyclical transcriptional activation and delayed negative feedback regulation is emerging as the fundamental mechanism underlying the segmentation clock. Ubiquitin-dependent protein degradation and probably other posttranslational regulations are also required. Fgf8 and Wnt3a gradients are important in positioning somite boundaries and, probably, in coordinating tail growth and segmentation. The circadian clock is another biochemical oscillator, which, similar to the segmentation clock, is operated with a negative transcription-regulated feedback mechanism. While the circadian clock uses a more complicated network of pathways to achieve homeostasis, it appears that the segmentation clock exploits the Notch pathway to achieve both signal generation and synchronization. We also discuss mathematical modeling and future directions in the end.

In vertebrates with mutations in the Notch cell-cell communication pathway, segmentation fails: the boundaries demarcating somites, the segments of the embryonic body axis, are absent or irregular. This phenotype has prompted many investigations, but the role of Notch signalling in somitogenesis remains mysterious. Somite patterning is thought to be governed by a "clock-and-wavefront" mechanism: a biochemical oscillator (the segmentation clock) operates in the cells of the presomitic mesoderm, the immature tissue from which the somites are sequentially produced, and a wavefront of maturation sweeps back through this tissue, arresting oscillation and initiating somite differentiation. Cells arrested in different phases of their cycle express different genes, defining the spatially periodic pattern of somites and controlling the physical process of segmentation. Notch signalling, one might think, must be necessary for oscillation, or to organize subsequent events that create the somite boundaries. Here we analyse a set of zebrafish mutants and arrive at a different interpretation: the essential function of Notch signalling in somite segmentation is to keep the oscillations of neighbouring presomitic mesoderm cells synchronized.

Four genes – deltaA, deltaB, deltaC and deltaD – coding for homologues of the Notch ligand Delta have been discovered in zebrafish ( Haddon et al., 1998b ). We report here the cDNA sequence and expression pattern of deltaC. Its closest relatives are deltaB and Xenopus X-Delta-2. Unlike deltaA, deltaB, and deltaD, deltaC is not expressed in the majority of nascent primary neurons; but it is strongly expressed in the early retina, where it precedes other delta genes. It is also expressed in cranial ganglia, in sensory epithelia including ear and lateral line, and in scattered epidermal cells. In the mesoderm, expression is visible by 50% epiboly; it is seen subsequently in the tail bud, in stripes in the presomitic mesoderm and in the posterior half of each somite. There is expression also in notochord, blood vessels and pronephros.

Mechanosensory hair cells in the sensory patches of the vertebrate ear are interspersed among supporting cells, forming a fine-grained pattern of alternating cell types. Analogies with Drosophila mechanosensory bristle development suggest that this pattern could be generated through lateral inhibition mediated by Notch signalling. In the zebrafish ear rudiment, homologues of Notch are widely expressed, while the Delta homologues deltaA, deltaB and deltaD, coding for Notch ligands, are expressed in small numbers of cells in regions where hair cells are soon to differentiate. This suggests that the delta-expressing cells are nascent hair cells, in agreement with findings for Delta1 in the chick. According to the lateral inhibition hypothesis, the nascent hair cells, by expressing Delta protein, would inhibit their neighbours from becoming hair cells, forcing them to be supporting cells instead. The zebrafish mind bomb mutant has abnormalities in the central nervous system, somites, and elsewhere, diagnostic of a failure of Delta-Notch signalling: in the CNS, it shows a neurogenic phenotype accompanied by misregulated delta gene expression. Similar misregulation of delta genes is seen in the ear, along with misregulation of a Serrate homologue, serrateB, coding for an alternative Notch ligand. Most dramatically, the sensory patches in the mind bomb ear consist solely of hair cells, which are produced in great excess and prematurely; at 36 hours post fertilization, there are more than ten times as many as normal, while supporting cells are absent. A twofold increase is seen in the number of otic neurons also. The findings are strong evidence that lateral inhibition mediated by Delta-Notch signalling controls the pattern of sensory cell differentiation in the ear.

Notch signaling pathway defines an evolutionarily conserved mechanism in cell-fate determination in a broad spectrum of developmental processes through local cell interactions. mind bomb (mib) encodes an E3 ubiquitin ligase that is involved in Notch activation through Delta ubiquitylation and internalization. To further dissect the function of Mib, two yeast two-hybrid screens for zebrafish Mib/Mib2-binding proteins with different strategies have been performed. 81 putative interesting proteins were discovered and classified into six groups: ubiquitin proteasome pathway, cytoskeleton, trafficking, replication/transcription/translation factors, cell signaling and others. Confirmed by coimmunoprecipitation (Co-IP), Mib interacted with four tested proteins: ubiquitin specific protease 1 (Usp1), ubiquitin specific protease 9 (Usp9), tumor-necrosis-factor-receptor-associated factor (TRAF)-binding domain (Trabid)/zinc finger, RAN-binding domain containing 1 (Zranb1) and hypoxia-inducible factor 1, alpha subunit inhibitor (Hif1an)/factor inhibiting HIF 1 (Fih-1). Usp1, Usp9, Trabid and Fih-1 also bound to zebrafish Mib2, a Mib homolog with similar structural domains and functions. Both Mib and Mib2 can ubiquitylate Trabid and Fih-1, indicating a potential regulating role of Mib and Mib2 on Trabid and Fih-1 and, furthermore, the possible involvement of Notch signaling in hypoxia-regulated differentiation, tumorigenesis and NF-κB pathway. Finally, functions of confirmed Mib/Mib2-interacting proteins are collated, summarized and hypothesized, which depicts a regulating network beyond Notch signaling.

The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand–receptor combinations that occur concurrently during development in zebrafish.

BACKGROUND: There are four cell lineages derived from intestinal stem cells that are located at the crypt and villus in the mammalian intestine the non-secretory absorptive enterocytes, and the secretory cells, which include mucous-secreting goblet cells, regulatory peptide-secreting enteroendocrine cells and antimicrobial peptide-secreting Paneth cells. Although fibroblast growth factor (Fgf) signaling is important for cell proliferation and differentiation in various tissues, its role in intestinal differentiation is less well understood. METHODOLOGY/PRINCIPAL FINDINGS: We used a loss of function approach to investigate the importance of Fgf signaling in intestinal cell differentiation in zebrafish; abnormal differentiation of goblet cells was observed when Fgf signaling was inhibited using SU5402 or in the Tg(hsp70ldnfgfr1-EGFP) transgenic line. We identified Fgfr2c as an important receptor for cell differentiation. The number of goblet cells and enteroendocrine cells was reduced in fgfr2c morphants. In addition to secretory cells, enterocyte differentiation was also disrupted in fgfr2c morphants. Furthermore, proliferating cells were increased in the morphants. Interestingly, the loss of fgfr2c expression repressed secretory cell differentiation and increased cell proliferation in the mib(ta52b) mutant that had defective Notch signaling. CONCLUSIONS/SIGNIFICANCE: In conclusion, we found that Fgfr2c signaling derived from mesenchymal cells is important for regulating the differentiation of zebrafish intestine epithelial cells by promoting cell cycle exit. The results of Fgfr2c knockdown in mib(ta52b) mutants indicated that Fgfr2c signaling is required for intestinal cell differentiation. These findings provide new evidences that Fgf signaling is required for the differentiation of intestinal cells in the zebrafish developing gut.

In the past three decades, the number of zebrafish laboratories has significantly increased in Taiwan. The Taiwan Zebrafish Core Facility (TZCF), a government-funded core facility, was launched to serve this growing community. The Core Facility was built on two sites, one located at the National Health Research Institutes (NHRI, called Taiwan Zebrafish Core Facility at NHRI or TZeNH) and the other is located at the Academia Sinica (Taiwan Zebrafish Core Facility at AS a.k.a. TZCAS). The total surface area of the TZCF is about 180 m(2) encompassing 2880 fish tanks. Each site has a separate quarantine room and centralized water recirculating systems, monitoring key water parameters. To prevent diseases, three main strategies have been implemented: (1) imported fish must be quarantined; (2) only bleached embryos are introduced into the main facilities; and (3) working practices were implemented to minimize pathogen transfer between stocks and facilities. Currently, there is no health program in place; however, a fourth measure for the health program, specific regular pathogen tests, is being planned. In March 2015, the TZCF at NHRI has been AAALAC accredited. It is our goal to ensure that we provide "disease-free" fish and embryos to the Taiwanese research community.

In order to unfold the function of genes, solely performing mRNA over-expression is not enough nowadays. Traditional protein expression experiments, such as Western blotting and immunohistochemical staining, could only provide researchers the changes of expression levels and/or location of their targets. To make a more strong and convincing statement about gene function, it is necessary to perform both "gain-of-function" and "loss-of-function" studies. Both assays can be performed easily by transfecting DNA plasmid and siRNA in cell culture system; while in zebrafish, mRNA and morpholino (MO) microinjection can serve similar purposes. It is common for the zebrafish community to carry out microinjection experiments to explore a gene function. Instead of making a single knockdown/over-expression of a gene, we foresee that more and more large-scale screens on certain protein families will be performed in the future. Here, based on our previous experience in zebrafish "loss-of-function" screening on deubiquitylating enzymes, we describe a general work flow, from morpholino designation, in situ hybridization, to data analysis, as a reference for researchers who may be interested in a similar screen.

In order to unfold the function of genes, solely performing mRNA over-expression is not enough nowadays. Traditional protein expression experiments, such as Western blotting and immunohistochemical staining, could only provide researchers the changes of expression levels and/or location of their targets. To make a more strong and convincing statement about gene function, it is necessary to perform both "gain-of-function" and "loss-of-function" studies. Both assays can be performed easily by transfecting DNA plasmid and siRNA in cell culture system; while in zebrafish, mRNA and morpholino (MO) microinjection can serve similar purposes. It is common for the zebrafish community to carry out microinjection experiments to explore a gene function. Instead of making a single knockdown/over-expression of a gene, we foresee that more and more large-scale screens on certain protein families will be performed in the future. Here, based on our previous experience in zebrafish "loss-of-function" screening on deubiquitylating enzymes, we describe a general work flow, from morpholino designation, in situ hybridization, to data analysis, as a reference for researchers who may be interested in a similar screen.

Zebrafish cops6 encodes a putative deubiquitylating enzyme (DUB) that belongs to the JAMM family. It consists of 297 amino acids and includes the Mov34/MPN/PAD-1 (PF01398) domain. Ubiquitylation is involved in many cellular processes and deconjugation of ubiquitin-modified substrates is important to maintain a sufficient amount of free ubiquitin in the cell. Here, we report our findings regarding the general function of the cops6 gene, as a continuation of our previous studies involving DUB knockdown screening. We have found that cops6 plays different roles in early embryonic development in the zebrafish, including dorsoventral patterning, convergent extension movement and brain formation. In addition, our findings indicate that cops6 plays an anti-apoptotic role during segmentation. Overall, the present study that consolidates our previous work on zebrafish DUB genes, corroborates the hypothesis of multi-functional roles for DUB genes during development.

Background Deconjugation of ubiquitin and/or ubiquitin-like modified protein substrates is essential to modulate protein-protein interactions and, thus, signaling processes in cells. Although deubiquitylating (deubiquitinating) enzymes (DUBs) play a key role in this process, however, their function and regulation remain insufficiently understood. The "loss-of-function" phenotype studies can provide important information to elucidate the gene function, and zebrafish is an excellent model for this goal. Results From an in silico genome-wide search, we found more than 90 putative DUBs encoded in the zebrafish genome belonging to six different subclasses. Out of them, 85 from five classical subclasses have been tested with morpholino (MO) knockdown experiments and 57 of them were found to be important in early development of zebrafish. These DUB morphants resulted in a complex and pleiotropic phenotype that, regardless of gene target, always affected the notochord. Based on the huC neuronal marker expression, we grouped them into five sets (groups I to V). Group I DUBs (otud7b, uchl3 and bap1) appear to be involved in the Notch signaling pathway based on the neuronal hyperplasia, while group IV DUBs (otud4, usp5, usp15 and usp25) play a critical role in dorsoventral patterning through the BMP pathway. Conclusion We have identified an exhaustive list of genes in the zebrafish genome belonging to the five established classes of DUBs. Additionally, we performed the corresponding MO knockdown experiments in zebrafish as well as functional studies for a subset of the predicted DUB genes. The screen results in this work will stimulate functional follow-up studies of potential DUB genes using the zebrafish model system.